Establishment Of HBV CccDNA Drugs Screening System And The Exploration Of Potential Targets For HBV CccDNA Degradation

With the widespread use of the prophylactic hepatitis B vaccine,the incidence of HBV infection has declined significantly,however there are still 240 million people with chronic hepatitis B virus(CHB)infection worldwide and 650 thousand person die from liver diseases related with CHB infection annually.Therefore,CHB infection is still a worldwide public problem.Once the hepatocyte is infected by HBV,the virus replicative intermediate termed covalently closed circular DNA(cccDNA)is formed in the nucleus.The intracellular cccDNA,with low-content but long half-life,is the template of all kinds of HBV RNAs.It is currently believed that the persistence of cccDNA in hepatocytes is the main cause for HBV chronic infection and recurrence after drug cessation.However,clinical drugs for CHB treatment including nucleos(t)ide analogues(NAs)and interferon alpha(IFNa)could effectively suppress HBV replication,while NAs have little effect on the existed cccDNA,and the treatment period is quite long thus there is risk of drug resistance;IFNa could reduce the intracellular cccDNA to some extent,while its side effects are severe which results in limited treatment objects.The development of cccDNA drugs has been limited by the lack of its drugs screening system.Therefore,this study seeks to establish a stable and reliable cccDNA drugs screening system and use this system to explore potential targets for cccDNA degradation.First,we used the modified Hirt method to extract intracellular protein-free DNA(PF-DNA)of the HBV-stabilized integrant cell line HepAD38,identified the cccDNA,and established a Southern blot detection method for cccDNA;Besides,cccDNA-specific primers and probe were designed for a qPCR detection system using diluted HBV plasmids as standard samples(Linear range:4.0×103-4.0×109 copies/mL,R2=0.990).HepG2-NTCP 2B1 cells that overexpressed the HBV receptor sodium taurocholate cotransporting polypeptide(NTCP)were used for HBV infection and the intracellular cccDNA was detected.HBV-infected HepG2-NTCP 2B1 cells were treated with tenofovir(TDF)and interferon alpha(IFNα2b),and secreted HBV antigens and viral DNA were detected.Both TDF and IFNα2b were found to effectively inhibit viral replication,however TDF had much less inhibitory effects on viral antigens and cccDNA than IFNa2b,which was similar to the changes of virological parameters in CHB patients treated with these drugs,indicating that the system could be used for cccDNA drug screening.We further analyzed the correlation between intracellular cccDNA and secreted viral antigens,HBV DNAs of HBV-infected HepG2-NTCP 2B1 cells.The secreted HBeAg and intracellular HBV DNAs were revealed to have the most significant linear correlations with HBV cccDNA(r=0.84,p<0.01;r=0.84,p<0.0001).Since the detection of intracellular HBV DNAs involved nucleic acid extraction,which was relatively complicated,and the quantitative detection of secreted HBeAg was quick,easy and suitable for high-throughput operation,besides it would not be interfered by the virus brought into the infection,thus it was a better choice as the surrogate marker for cccDNA.Therefore,a stable and reliable cccDNA drugs screening system was established based on the HepG2-NTCP 2B1 cell model.We further found that cell passage and cryopreservation would reduce intracellular and extracellular virological indicators of HBV-infected HepG2-NTCP 2B1 to a certain extent,but it had little effect on the results of drug screening,which could shorten the experimental period.We further sought potential targets for HBV cccDNA degradation by this system.According to the literature research,four host genes(Apobec3A,Apobec3B,ISG15 and ISG20)that might be involved in HBV infection were targeted.HepG2-NTCP 2B1 cells with or without(w/o)HBV infection were treated with different concentrations of IFNa2b,and ISG15 and ISG20 were found to be specifically and dose-dependently upregulated by IFNa2b.Similarly,these two proteins but not Apobec3 A and Apobec3B were upregulated by IFNa2b in HepG2,Huh7,HepaRG and the modified HepaRG-M14a cells.Lentiviruses carrying these genes and adenoviruses expressing shRNA targeting the corresponding genes were constructed and four genes were overexpressed and down-regulated in HBV-infected HepG2-NTCP 2B1 to see their effects on HBV infection and cccDNA,By detecting HBV antigens and cccDNA,it was determined that ISG15 and ISG20,which were specifically stimulated by interferon,could degrade cccDNA.In summary,we have established a stable and reliable HBV cccDNA drugs screening system and verified that IFNa2b could effectively reduce cccDNA by this system.Meanwhile,we found that two host factors ISG15 and ISG20 stimulated specifically by IFNa2b could degrade HBV cccDNA.